利用人類臍帶靜脈內皮細胞探討 MT1-MMP在LPLs刺激的血管新生中可能的角色

Abstract

Sphingosine 1-phosphate (S1P) 和Lysophosphatidic acid (LPA) 是小分子量的水解磷酸質(lysophospholipids, LPLs)。 藉由接合到專一的G-protein couple受器，LPLs 調控許多細胞功能，如細胞增生、細胞遷移、細胞侵入、以及細胞的分化作用。基質金屬蛋白酶(matrix-metalloproteinases, MMPs)是一群需要鋅離子的蛋白酶，目前已經在人類體內發現了二十種以上的MMPs。MMPs藉由分解不同的細胞基質(extracellular matrix, ECM)調控細胞與細胞基質間的交互作用。膜型基質蛋白酶-1 (membrane Type 1-metalloproteinase, MT1-MMP)不只分解ECM蛋白 ，也幫助活化對細胞遷移非常重要的基質金屬蛋白酶-2 (metalloproteinase-2, MMP-2)。另外，基質金屬蛋白酶體內抑制劑-2 (tissue inhibitor of metalloproteinase-2, TIMP-2) 也會幫助活化MMP-2。在我們過去的研究中，發現LPLs會促進MMP-2在人類臍帶靜脈內皮細胞(human umbilical vein endothelial cells, HUVECs)中的表現。在本研究中，利用曠時聚合酶連鎖反應系統(real-time PCR)以及化學抑制劑，我們發現LPLs經由Gi- and Gq- 引導的途徑來促進MT1-MMP以及TIMP-2的表現。接下來，我們利用MT1-MMP siRNA的方式來降低細胞內MT1-MMP的表現探討MT1-MMP在細胞功能中扮演的角色。在此，我們發現MT1-MMP的剔減(knock down)會使MMP-2活化作用、細胞貼附(cell adhesion)、管形構造形成(tube formation)作用被抑制。另外，我們發現MMP-2以及TIMP-2的表現也會因為MT1-MMP siRNA的導入受到抑制。總結來說，藉由調控這些分子以及生理現象，MT1-MMP在LPLs調控的血管新生中可能扮演著極重要的角色。

Sphingosine 1-phosphate (S1P) and Lysophosphatidic acid (LPA) are low molecular weight lysophospholipids (LPLs). Through binding to specific G protein-coupled receptor family, LPLs regulate various cellular functions, including proliferation, migration, invasion, and differentiation. Matrix-Metalloproteinases (MMPs) are Zinc-dependent proteases and more than twenty MMPs have been identified in human. MMPs play important roles in modulating interactions between cells and extracellular matrix (ECM) via the degradation of various matrix proteins. Membrane type 1-metalloproteinase (MT1-MMP) not only degrades ECM protein but also activates metalloproteinase-2 (MMP-2, Gelatinase A), which is important to endothelial cell migration. In addition, tissue inhibitor of metalloproteinase-2 (TIMP-2) also regulates MMP-2 activation. Our previous study showed that LPLs enhance MMP-2 expression and activity in human umbilical vein endothelial cells (HUVECs). In this study, we revealed that LPLs also induce MT1-MMP and TIMP-2 expression in HUVECs through real-time PCR. Furthermore, in the presence of chemical inhibitors, we demonstrated that LPLs-induced MT1-MMP and TIMP-2 expression are through the Gi- and Gq- dependent pathways. Next, we studied the function of MT1-MMP by introducing MT1-MMP siRNA. Herein, we clarified that MT1-MMP is involved in S1P-induced endothelial cell (EC) functions by conducting the MMP-2 activity assay, EC adhesion, and tube formation assay. In the last part, we found that MT1-MMP siRNA down-regulates MMP-2 and TIMP-2 expressions. This indicates that MT1-MMP not only regulates MMP-2 activity but also modulates the expression of MMP-2. To sum up, we suggest that LPLs play a role in regulating MT1-MMP and TIMP-2 expression; moreover, through regulating MMP-2 activity and expression, EC adhesion, and tube formation, MT1-MMP may be an important mediator of LPLs-induced angiogenesis.